Absence Of Extracellular Cl Research Articles

Substrate recognition of renally eliminated angiotensin II receptor blockers by organic anion transporter 4

Organic anion transporter (OAT) 4, which is localized at the apical membrane of human renal proximal tubules, transports olmesartan, an angiotensin II receptor blocker (ARB). Many ARBs, including olmesartan, undergo partial tubular secretion as active forms, and inhibit OAT4-mediated uptake activity. Here, we examined the substrate recognition of various ARBs by OAT4 in order to assess whether OAT4 might be involved in the renal handling of ARBs. Concentration-dependent OAT4-mediated uptake of azilsartan, candesartan, carboxylosartan, losartan, and valsartan was observed with Km values of 6.6, 31, 7.2, 13, and 1.7 μM, respectively, in the absence of extracellular Cl−. In the presence of extracellular Cl−, OAT4-mediated uptake of dianionic ARBs (azilsartan, candesartan, carboxylosartan, and valsartan) was lower and reached a steady state faster than in the absence of extracellular Cl−. Thus, OAT4 is proposed to use extracellular Cl− as a counterpart for anion efflux. Our results suggest that OAT4 may play a role in the excretion of azilsartan, candesartan, carboxylosartan, and valsartan, as well as olmesartan. In contrast, OAT4-mediated uptake of losartan, a monoanionic ARB, was little affected by extracellular Cl−, suggesting that only OAT4-mediated dianion transport is Cl−-sensitive.

Molecular Basis and Differentiation-Associated Alterations of Anion Secretion in Human Duodenal Enteroid Monolayers

Background & AimsHuman enteroids present a novel tool to study human intestinal ion transport physiology and pathophysiology. The present study describes the contributions of Cl- and HCO3- secretion to total cyclic adenosine monophosphate (cAMP)-stimulated electrogenic anion secretion in human duodenal enteroid monolayers and the relevant changes after differentiation.MethodsHuman duodenal enteroids derived from 4 donors were grown as monolayers and differentiated by a protocol that includes the removal of Wnt3A, R-spondin1, and SB202190 for 5 days. The messenger RNA level and protein expression of selected ion transporters and carbonic anhydrase isoforms were determined by quantitative real-time polymerase chain reaction and immunoblotting, respectively. Undifferentiated and differentiated enteroid monolayers were mounted in the Ussing chamber/voltage-current clamp apparatus, using solutions that contained as well as lacked Cl- and HCO3-/CO2, to determine the magnitude of forskolin-induced short-circuit current change and its sensitivity to specific inhibitors that target selected ion transporters and carbonic anhydrase(s).ResultsDifferentiation resulted in a significant reduction in the messenger RNA level and protein expression of cystic fibrosis transmembrane conductance regulator, (CFTR) Na+/K+/2Cl- co-transporter 1 (NKCC1), and potassium channel, voltage gated, subfamily E, regulatory subunit 3 (KCNE3); and, conversely, increase of down-regulated-in-adenoma (DRA), electrogenic Na+/HCO3- co-transporter 1 (NBCe1), carbonic anhydrase 2 (CA2), and carbonic anhydrase 4 (CA4). Both undifferentiated and differentiated enteroids showed active cAMP-stimulated anion secretion that included both Cl- and HCO3- secretion as the magnitude of total active anion secretion was reduced after the removal of extracellular Cl- or HCO3-/CO2. The magnitude of total anion secretion in differentiated enteroids was approximately 33% of that in undifferentiated enteroids, primarily owing to the reduction in Cl- secretion with no significant change in HCO3- secretion. Anion secretion was consistently lower but detectable in differentiated enteroids compared with undifferentiated enteroids in the absence of extracellular Cl- or HCO3-/CO2. Inhibiting CFTR, NKCC1, carbonic anhydrase(s), cAMP-activated K+ channel(s), and Na+/K+-adenosine triphosphatase reduced cAMP-stimulated anion secretion in both undifferentiated and differentiated enteroids.ConclusionsHuman enteroids recapitulate anion secretion physiology of small intestinal epithelium. Enteroid differentiation is associated with significant alterations in the expression of several ion transporters and carbonic anhydrase isoforms, leading to a reduced but preserved anion secretory phenotype owing to markedly reduced Cl- secretion but no significant change in HCO3- secretion.

Cellular Uptake of Levocetirizine by Organic Anion Transporter 4

The pharmacokinetics of cetirizine, a nonsedating antihistamine, is profoundly affected by transporter-mediated membrane transport in the kidney. In this study, we aimed to investigate the transport mechanism of levocetirizine, the pharmacologically active enantiomer of cetirizine, via human organic anion transporter 4 (OAT4) expressed in the apical membrane of renal proximal tubules and the basal plasma membrane of placental syncytiotrophoblasts. In cells expressing human OAT4 under the control of tetracycline, levocetirizine uptake was increased by tetracycline treatment. On the other hand, OAT4 expression did not facilitate efflux of preloaded levocetirizine from the cells, either in the presence or absence of extracellular Cl−. The OAT4-mediated levocetirizine uptake was concentration-dependent with a Km of 38 μM. The uptake rate of levocetirizine via OAT4 was approximately twice that of racemic cetirizine, indicating stereoselective uptake of levocetirizine. On the other hand, OAT4-mediated [3H]dehydroepiandrosterone sulfate uptake was inhibited by dextrocetirizine and levocetirizine. Overall, our findings indicate that OAT4 mediates levocetirizine uptake but is unlikely to mediate the efflux.

Influences of Chloride and Hypochlorite on Neutrophil Extracellular Trap Formation

BackgroundThe release by neutrophils of DNA-based extracellular traps (NETs) is a recently recognized innate immune phenomenon that contributes significantly to control of bacterial pathogens at tissue foci of infection. NETs have also been implicated in the pathogenesis of non-infectious diseases such as small vessel vasculitis, lupus and cystic fibrosis lung disease. Reactive oxygen species (ROS) are important mediators of NET generation (NETosis). Neutrophils with reduced ROS production, such as those from patients with chronic granulomatous disease or myeloperoxidase (MPO) deficiency, produce fewer NETs in response to inflammatory stimuli. To better understand the roles of various ROS in NETosis, we explore the role of MPO, its substrates chloride ion (Cl−) and hydrogen peroxide (H2O2), and its product hypochlorite (HOCl) in NETosis.FindingsIn human peripheral blood neutrophils, pharmacologic inhibition of MPO decreased NETosis. Absence of extracellular Cl−, a substrate for MPO, also reduced NETosis. While exogenous addition of H2O2 and HOCl stimulated NETosis, only exogenous HOCl could rescue NETosis in the setting of MPO inhibition. Neither pharmacological inhibition nor genetic deletion of MPO in murine neutrophils blocked NETosis, in contrast to findings in human neutrophils.ConclusionsOur results pinpoint HOCl as the key ROS involved in human NETosis. This finding has implications for understanding innate immune function in diseases in which Cl− homeostasis is disturbed, such as cystic fibrosis. Our results also reveal an example of significant species-specific differences in NET phenotypes, and the need for caution in extrapolation to humans from studies of murine NETosis.

Na+-dependent HCO3− Import by the slc4a10 Gene Product Involves Cl− Export

The slc4a10 gene encodes an electroneutral Na(+)-dependent HCO(3)(-) importer for which the precise mode of action remains unsettled. To resolve this issue, intracellular pH (pH(i)) recordings were performed upon acidification in the presence of CO(2)/HCO(3)(-) by 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) fluorometry of stably slc4a10-transfected NIH-3T3 fibroblasts. slc4a10 expression induced a significant Na(+)-dependent pH(i) recovery, which was accompanied by an increase in the intracellular Na(+) concentration evaluated by use of the Na(+)-sensitive fluorophore CoroNa Green. The estimated Na(+):HCO(3)(-) stoichiometry was 1:2. Cl(-) is most likely the counterion maintaining electroneutrality because (i) Na(+)-dependent pH(i) recovery was eliminated in Cl(-)-depleted cells; (ii) acute extracellular Cl(-) removal led to a larger alkalization in slc4a10-transfected cells than in control cells; and (iii) the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS)-sensitive and Na(+)- and HCO(3)(-)-dependent (36)Cl(-)-efflux during pH(i) recovery was significantly greater in acidified slc4a10-transfected cells than in control cells. Charged amino acids specific to slc4a gene family members that transport Na(+) and are expected to move more HCO(3)(-) molecules/turnover were targeted by site-directed mutagenesis. Na(+)-dependent pH(i) recovery was reduced in each of the single amino acid mutated cell lines (E890A, E892A, H976L, and H980G) compared with wild type slc4a10-transfected cells and completely eliminated in quadruple mutant cells. In conclusion, the data suggest that slc4a10 expressed in mammalian cells encodes a Na(+)-dependent Cl(-)/HCO(3)(-) exchanger in which four specific charged amino acids seem necessary for ion transport.

Characterization of Cellular Uptake of Perfluorooctanoate via Organic Anion-Transporting Polypeptide 1A2, Organic Anion Transporter 4, and Urate Transporter 1 for Their Potential Roles in Mediating Human Renal Reabsorption of Perfluorocarboxylates

It has been hypothesized that human renal apical membrane transporters play a key role in human renal reabsorption of perfluorooctanoate (PFO), which contributes to the long half-life of PFO in humans. In the present study, PFO uptake kinetics of human organic anion-transporting polypeptide (OATP) 1A2, organic anion transporter (OAT) 4, and urate transporter 1 (URAT1) in stably transfected cell lines was investigated. OAT4 and URAT1, but not OATP1A2, were shown to mediate saturable PFO cellular uptake. OAT4-mediated PFO uptake was stimulated by a low extracellular pH, which was evidenced as a lower Michaelis constant (K(m)) at pH 6 (172.3 ± 45.9μM) than that at pH 7.4 (310.3 ± 30.2μM). URAT1-mediated PFO uptake was greatly enhanced by an outward Cl(-) gradient, and its K(m) value was determined to be 64.1 ± 30.5μM in the absence of extracellular Cl(-). The inhibition of OATP1A2- or OAT4-mediated estrone-3-sulfate uptake or URAT1-mediated urate uptake has been compared for linear perfluorocarboxylates (PFCs) with carbon chain lengths from 4 to 12. A clear chain length-dependent inhibition was observed, suggesting that PFCs in general are substrates of OAT4 and URAT1 but with different levels of affinities to the transporters depending on their chain length. Our results suggest that OAT4 and URAT1 are key transporters in renal reabsorption of PFCs in humans and, as a result, may contribute significantly to the long half-life of PFO in humans.

Extracellular Chloride Regulates the Epithelial Sodium Channel

The extracellular domain of the epithelial sodium channel ENaC is exposed to a wide range of Cl(-) concentrations in the kidney and in other epithelia. We tested whether Cl(-) alters ENaC activity. In Xenopus oocytes expressing human ENaC, replacement of Cl(-) with SO4(2-), H2PO4(-), or SCN(-) produced a large increase in ENaC current, indicating that extracellular Cl(-) inhibits ENaC. Extracellular Cl(-) also inhibited ENaC in Na+-transporting epithelia. The anion selectivity sequence was SCN(-) < SO4(2-) < H2PO4(-) < F(-) < I(-) < Cl(-) < Br(-). Crystallization of ASIC1a revealed a Cl(-) binding site in the extracellular domain. We found that mutation of corresponding residues in ENaC (alpha(H418A) and beta(R388A)) disrupted the response to Cl(-), suggesting that Cl(-) might regulate ENaC through an analogous binding site. Maneuvers that lock ENaC in an open state (a DEG mutation and trypsin) abolished ENaC regulation by Cl(-). The response to Cl(-) was also modulated by changes in extracellular pH; acidic pH increased and alkaline pH reduced ENaC inhibition by Cl(-). Cl(-) regulated ENaC activity in part through enhanced Na+ self-inhibition, a process by which extracellular Na+ inhibits ENaC. Together, the data indicate that extracellular Cl(-) regulates ENaC activity, providing a potential mechanism by which changes in extracellular Cl(-) might modulate epithelial Na+ absorption.

Nitric Oxide Transiently Converts Synaptic Inhibition to Excitation in Retinal Amacrine Cells

Nitric oxide (NO) is generated by multiple cell types in the vertebrate retina, including amacrine cells. We investigate the role of NO in the modulation of synaptic function using a culture system containing identified retinal amacrine cells. We find that moderate concentrations of NO alter GABA(A) receptor function to produce an enhancement of the GABA-gated current. Higher concentrations of NO also enhance GABA-gated currents, but this enhancement is primarily due to a substantial positive shift in the reversal potential of the current. Several pieces of evidence, including a similar effect on glycine-gated currents, indicate that the positive shift is due to an increase in cytosolic Cl-. This change in the chloride distribution is especially significant because it can invert the sign of GABA- and glycine-gated voltage responses. Furthermore, current- and voltage-clamp recordings from synaptic pairs of GABAergic amacrine cells demonstrate that NO transiently converts signaling at GABAergic synapses from inhibition to excitation. Persistence of the NO-induced shift in E(Cl-) in the absence of extracellular Cl- indicates that the increase in cytosolic Cl- is due to release of Cl- from an internal store. An NO-dependent release of Cl- from an internal store is also demonstrated for rat hippocampal neurons indicating that this mechanism is not restricted to the avian retina. Thus signaling in the CNS can be fundamentally altered by an NO-dependent mobilization of an internal Cl- store.

PGE2 stimulates Cl- secretion in murine M-1 cortical collecting duct cells in an autocrine manner.

Prostaglandin E2 (PGE2) is thought to be an important modulator of renal ion and water transport, but its effects remain complex and incompletely understood. Here we examined the effects of PGE2 on transepithelial ion transport of M-1 mouse cortical collecting duct cells using short-circuit current (ISC) measurements. Basolateral addition of PGE2 (1 microM) produced a transient peak increase in ISC of 6.3+/-0.8 microA cm(-2) (n=11), followed by a sustained plateau. The PGE2-evoked response was preserved in the presence of 100 micro M apical amiloride with an average peak increase of 10.6+/-1.0 microA cm(-2) (n=23). However, it was greatly diminished in both the presence of apical diphenylamine-2-carboxylic acid (DPC, 1 mM) and the absence of extracellular Cl-, indicating that Cl- secretion had been stimulated. Basolateral PGE2 induced a concentration dependent response, with an EC50 of about 8 nM. Apical addition of PGE2 elicited an ISC response similar to that observed with basolateral PGE2. Furthermore, apical exposure to arachidonic acid (AA) produced a similar increase in ISC, which could be prevented by the cyclooxygenase inhibitor indomethacin, while AA failed to exert an additional effect in the presence of PGE2. Using RT-PCR, we confirmed the expression of the PGE2 (EP) receptor subtypes EP1, EP3 and EP4 but not of EP2 in cultured M-1 CCD cells. We conclude that M-1 cells express functional cyclooxygenase activity and can generate PGE2 which acts in an autocrine manner, causing Cl- secretion.

Basolateral adrenoceptor activation mediates noradrenaline-induced Cl- secretion in M-1 mouse cortical collecting duct cells.

Noradrenaline (NA) released from efferent renal sympathetic nerves may directly affect renal tubular transport. Here we examined the effect of NA on transepithelial ion transport of cultured M-1 mouse cortical collecting duct cells using equivalent short-circuit current ( I(SC)) measurements. Steady-state I(SC) averaged 87.5+/-2.9 microA cm(-2) (n=185) and was reduced by 97.1+/-0.1% (n=80) by apical amiloride (100 microM) confirming that the predominant electrogenic transport across M-1 monolayers is sodium absorption via the epithelial sodium channel (ENaC). Basolateral addition of NA (10 microM) induced a biphasic change in I(SC) characterized by an initial transient peak increase of 18.1+/-0.9 microA cm(-2) with a subsequent decline to a plateau 1.4+/-0.3 microA cm(-2) (n=20) above baseline. Apical application of NA had no effect. The response to basolateral NA was concentration dependent and was preserved in the presence of apical amiloride. In contrast, the response was largely reduced in the presence of apical diphenylamine-2-carboxylic acid (1 mM) and in the absence of extracellular Cl(-). The peak response to NA was reduced in the presence of the alpha-adrenoceptor antagonist phentolamine (100 microM), whereas the beta-antagonist propranolol (100 microM) reduced the secondary plateau phase while failing to influence the peak response. The alpha(1)-adrenoceptor-selective antagonists prazosin (10 nM) and corynanthine (1 microM) reduced the NA-induced peak response by about 75% and 70%, respectively, while the alpha(2)-adrenoceptor antagonist yohimbine (100 nM) was ineffective. We conclude that in M-1 cells NA stimulates Cl(-) secretion probably involving both alpha(1)- and beta-adrenoceptors located basolaterally.

Basolateral PAR-2 receptors mediate KCl secretion and inhibition of Na+ absorption in the mouse distal colon.

Proteinase-activated receptor-2 (PAR-2) may participate in epithelial ion transport regulation. Here we examined the effect of mouse activating peptide (mAP), a specific activator of PAR-2, on electrogenic transport of mouse distal colon using short-circuit current (I(SC)) measurements. Under steady-state conditions, apical application of amiloride (100 microM) revealed a positive I(SC) component of 74.3 +/- 6.8 microA x cm(-2) indicating the presence of Na+ absorption, while apical Ba2+ (10 mM) identified a negative I(SC) component of 26.2 +/- 1.8 microA x cm(-2) consistent with K+ secretion. Baseline Cl- secretion was minimal. Basolateral addition of 20 microM mAP produced a biphasic I(SC) response with an initial transient peak increase of 11.2 +/- 0.9 microA x cm(-2), followed by a sustained fall to a level 31.2 +/- 2.6 microA x cm(-2) (n = 43) below resting I(SC). The peak response was due to Cl- secretion as it was preserved in the presence of amiloride but was largely reduced in the presence of basolateral bumetanide (20 microM) or in the absence of extracellular Cl-. The secondary decline of I(SC) was also attenuated by bumetanide and by Ba2+, indicating that it is partly due to a stimulation of K+ secretion. In addition, the amiloride-sensitive I(SC) was slightly reduced by mAP, suggesting that inhibition of Na+ absorption also contributes to the I(SC) decline. Expression of PAR-2 in mouse distal colon was confirmed using RT-PCR and immunocytochemistry. We conclude that functional basolateral PAR-2 is present in mouse distal colon and that its activation stimulates Cl- and K+ secretion while inhibiting baseline Na+ absorption.

Electrophysiology of betaine transport in isolated perfused straight proximal tubule.

Betaine is accumulated in proximal renal tubular cells as an organic osmolyte. In theory, concentrative cellular uptake of betaine can be accomplished by the Na+, Cl-, betaine, GABA-cotransport system (BGT-1) cloned from MDCK cells. The carrier mediates the Na(+)-coupled electrogenic transport of organic osmolytes. Cl- may be transported together with Na+ and organic substrate but it is not an obligatory substrate. The expression of BGT is upregulated by osmotic cell shrinkage. In the present study, isolated perfused straight proximal tubules of the mouse were studied using electrophysiological techniques to elucidate the effects of betaine and gamma-aminobutyric acid (GABA) on the potential difference across the basolateral cell membrane (PDbl). Betaine and to a lesser extent GABA added to the bath led to the rapid depolarization of PDbl. The betaine-induced depolarization was virtually abolished in the absence of extracellular Na+ and blunted in the absence of extracellular Cl-. Inhibition of K+ channels by Ba2+ did not significantly modify betaine-induced depolarization. Increases of extracellular osmolarity enhanced, and decreases of extracellular osmolarity blunted, the betaine-induced depolarization. In conclusion, betaine transport across the basolateral cell membrane of straight proximal tubules from the mouse kidney is similar to the transport by BGT in terms of Na+ and Cl- sensitivity but not the apparent affinity for GABA. The sensitivity to ambient osmolarity indicates that betaine transport in proximal tubules is regulated rapidly and nongenomically.

ATP stimulates Cl- secretion and reduces amiloride-sensitive Na+ absorption in M-1 mouse cortical collecting duct cells.

1. Using equivalent short circuit current (ISC) measurements we examined the effect of extracellular ATP on transepithelial ion transport in M-1 mouse cortical collecting duct cells. Apical addition of ATP produced a rapid transient peak increase in ISC. This was followed by a fall below basal ISC due to a reduction in the amiloride-sensitive ISC component. 2. The ATP-induced ISC increase was preserved in the presence of apical amiloride while it was reduced in the absence of extracellular Cl- and in the presence of the apical Cl- channel blockers diphenylamine-2-carboxylic acid (DPC, 1 mM), DIDS (300 microM) and niflumic acid (100 microM). 3. The stimulatory effect of apical ATP on ISC was concentration dependent with an EC50 of about 0.6 microM. Basolateral ATP elicited a similar ISC response. Experiments using the ATP scavenger hexokinase demonstrated that the ATP effects were elicited via separate apical and basolateral receptors. 4. ATP and UTP applied to either the apical or the basolateral bath equi-potently stimulated ISC while 'purified' ADP and UDP had no effect consistent with P2Y2 purinoceptors, the expression of which was confirmed using RT-PCR. 5. Intracellular calcium concentration ([Ca2+]i) measurements using fura-2 demonstrated that ATP and UTP elicited a rise in [Ca2+]i with EC50 values of 1.1 and 0.6 microM, respectively. The shape and time course of the calcium response were similar to those of the ISC response. The peak ISC response was preserved in the nominal absence of extracellular calcium but was significantly reduced in cells pre-incubated with the calcium chelator BAPTA AM. 6. We conclude that in M-1 cells extracellular ATP reduces amiloride-sensitive Na+ absorption and stimulates Cl- secretion via calcium-activated Cl- channels through activation of P2Y2 purinoreceptors located in the apical and basolateral membrane.

Basolateral proteinase-activated receptor (PAR-2) induces chloride secretion in M-1 mouse renal cortical collecting duct cells.

1. Using RT-PCR, Northern blot analysis, and immunocytochemistry, we confirmed renal expression of proteinase-activated receptor (PAR-2) and demonstrated its presence in native renal epithelial and in cultured M-1 mouse cortical collecting duct (CCD) cells. 2. We investigated the effects of a PAR-2 activating peptide (AP), corresponding to the tethered ligand that is exposed upon trypsin cleavage, and of trypsin on M-1 cells using patch-clamp, intracellular calcium (fura-2) and transepithelial short-circuit current (ISC) measurements. 3. In single M-1 cells, addition of AP elicited a concentration-dependent transient increase in the whole-cell conductance. Removal of extracellular Na+ had no effect while removal of Cl- prevented the stimulation of outward currents. The intracellular calcium concentration increased significantly upon application of AP while a Ca2+-free pipette solution completely abolished the electrical response to AP. 4. In confluent monolayers of M-1 cells, apical application of AP had no effect on ISC whereas subsequent basolateral application elicited a transient increase in ISC. This increase was not due to a stimulation of electrogenic Na+ absorption since the response was preserved in the presence of amiloride. 5. The ISC response to AP was reduced in the presence of the Cl- channel blocker diphenylamine-2-carboxylic acid on the apical side and abolished in the absence of extracellular Cl-. 6. Trypsin elicited similar responses to those to AP while application of a peptide (RP) with the reverse amino acid sequence of AP had no effect on whole-cell currents or ISC. 7. In conclusion, our data suggest that AP or trypsin stimulates Cl- secretion by Ca2+-activated Cl- channels in M-1 CCD cells by activating basolateral PAR-2.

Mechanisms of pHi regulation studied in individual neurons cultured from mouse cerebral cortex.

Maintenance and regulation of intracellular pH (pHi) was studied in single cultured mouse neocortical neurons using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Reversal of the Na+ gradient by reduction of the extracellular Na+ concentration ([Na+]o) resulted in rapid intracellular acidification, inhibited by 5'-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na+/H+ exchange. In the presence of EIPA and/or 4',4'-diisothiocyano-stilbene-2',2'-sulfonic acid (DIDS), an inhibitor of Na+-coupled anion exchangers and Na+-HCO3- cotransport, a slow decline of pHi was seen. Following intracellular acidification imposed by an NH4Cl prepulse, pHi recovered at a rapid rate, which was reduced by reduction of [Na+]o and was virtually abolished by EIPA and DIDS in combination. Creating an outward Cl- gradient by removal of extracellular Cl- significantly increased the rate of pHi recovery. In HCO3(-)-free media, the pHi recovery rate was reduced in control cells and was abolished at zero [Na+]o and by EIPA. After intracellular alkalinization imposed by an acetate prepulse, pHi recovery was unaffected by DIDS but was significantly reduced in the absence of extracellular Cl-, as well as in the presence of Zn2+, which is a blocker of proton channels. Together, this points toward a combined role of DIDS-insensitive Cl-/HCO3- and passive H+ influx in the recovery of pHi after alkalinization.

Mechanisms of pHi regulation studied in individual neurons cultured from mouse cerebral cortex

Maintenance and regulation of intracellular pH (pHi) was studied in single cultured mouse neocortical neurons using the fluorescent probe 2′,7′-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). Reversal of the Na+ gradient by reduction of the extracellular Na+ concentration ([Na+]o) resulted in rapid intracellular acidification, inhibited by 5′-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na+/H+ exchange. In the presence of EIPA and/or 4′,4′-diisothiocyano-stilbene-2′,2′-sulfonic acid (DIDS), an inhibitor of Na+-coupled anion exchangers and Na+-HCO3− cotransport, a slow decline of pHi was seen. Following intracellular acidification imposed by an NH4Cl prepulse, pHi recovered at a rapid rate, which was reduced by reduction of [Na+]o and was virtually abolished by EIPA and DIDS in combination. Creating an outward Cl− gradient by removal of extracellular Cl− significantly increased the rate of pHi recovery. In HCO3−-free media, the pHi recovery rate was reduced in control cells and was abolished at zero [Na+]o and by EIPA. After intracellular alkalinization imposed by an acetate prepulse, pHi recovery was unaffected by DIDS but was significantly reduced in the absence of extracellular Cl−, as well as in the presence of Zn2+, which is a blocker of proton channels. Together, this points toward a combined role of DIDS-insensitive Cl−/HCO3− and passive H+ influx in the recovery of pHi after alkalinization. J. Neurosci. Res. 51:431–441, 1998. © 1998 Wiley-Liss, Inc.

Organic Anion Transporting Polypeptide Mediates Organic Anion/HCO3− Exchange

Organic anion transporting polypeptide (oatp) is an integral membrane protein cloned from rat liver that mediates Na+-independent transport of organic anions such as sulfobromophthalein and taurocholic acid. Previous studies in rat hepatocytes suggested that organic anion uptake is associated with base exchange. To better characterize the mechanism of oatp-mediated organic anion uptake, we examined transport of taurocholate in a HeLa cell line stably transfected with oatp under the regulation of a zinc-inducible promoter (Shi, X., Bai, S., Ford, A. C., Burk, R. D., Jacquemin, E., Hagenbuch, B., Meier, P. J., and Wolkoff, A. W. (1995) J. Biol. Chem. 270, 25591-25595). Whereas noninduced transfected cells showed virtually no uptake of [3H]taurocholate, taurocholate uptake by induced cells was Na+-independent and saturable (Km = 19.4 +/- 3.3 microM; Vmax = 62.2 +/- 1.4 pmol/min/mg protein; n = 3). To test whether organic anion transport is coupled to HCO3- extrusion, we compared the rates of taurocholate-dependent HCO3- efflux from alkali-loaded noninduced and induced cells. Monolayers grown on glass coverslips were loaded with the pH-sensitive dye 2', 7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; intracellular pH (pHi) was measured by excitation ratio fluorometry. Noninduced and induced cells were alkalinized to an equivalent pHi ( approximately 7.7) by transient exposure to a 50 mM HCO3-, Cl--free solution. In the absence of extracellular Cl- and taurocholate, isohydric reduction of superfusate HCO3- concentration from 50 to 25 mM resulted in an insignificant change in pHi over time (dpHi/dt) in both groups. Addition of 25 microM taurocholate to the superfusate led to a rapid fall in pHi in induced (-0.037 +/- 0.011 pH units/min to pHi of 7.41 +/- 0.14) but not in noninduced (0.003 +/- 0.006 pH units/min to pHi of 7.61 +/- 0.08) cells (p < 0.03). These data indicate that oatp-mediated taurocholate transport is Na+-independent, saturable, and accompanied by HCO3- exchange. We conclude that organic anion/base exchange is an important, potentially regulatable component of oatp function.

Mechanisms of pHi recovery from NH4Cl-induced acidosis in anoxic isolated turtle heart: a 31P-NMR study.

Mechanisms of intracellular pH (pHi) recovery from NH4Cl-induced acidosis were investigated on isolated perfused hearts of the turtle, Chrysemys picta bellii, using 31P nuclear magnetic resonance (NMR) spectroscopy at 20 degrees C. A major goal was to assess the activity of these mechanisms under anoxic conditions. Based on calculated buffer capacity and a pHi recovery range at 20 degrees C of 6.75-6.95 (normal pHi 7.2-7.4), mean H' efflux rate during perfusion with CO2-free N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)-buffered Ringer was only 15% (normoxia) and 25% (anoxia) of that with HCO3-buffered Ringer. With HCO3 solution, anoxic H1 efflux rate was approximately 50% of normoxia (0.333 vs. 0.645 mmol.l-1.min-1), but in TES solution, H1 efflux rate was unaffected by anoxia. To further characterize the transporters, we used blockers [the Na(+)-H+ antiport inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and the anion exchanger inhibitor 4,4'diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS)], ion substitution, and temperature change. EIPA (10 microM) inhibited H+ efflux rate by 40% in anoxic TES solution; DIDS (0.5 mM) blocked H+ efflux rate by 85% in anoxic HCO3 solution. No pHi recovery was observed in either normoxic or anoxic Na(+)-free solutions, but normal recovery was observed in the absence of extracellular Cl-. Recovery of pHi occurred 2-3 times faster at 30 degrees C than at 20 degrees C. ATP was unaffected by any manipulation in this study, whereas creatine phosphate (CP) fell during anoxia, and both CP and mechanical performance changed in parallel to pHi. We conclude that pHi regulation functions during anoxia, although at a reduced rate, and that recovery from acidosis is dominated, during both normoxia and anoxia, by a DIDS-sensitive Na+ and HCO3(-)-dependent mechanism, whereas EIPA-sensitive Na(+)-H+ antiport plays a less important role.

Chloride is required for receptor-mediated divalent cation entry in mesangial cells.

Agonists which stimulate the inositol 1,4,5 trisphosphate ([1,4,5]-IP3)-dependent mobilization of Ca2+ from intracellular stores also stimulate entry of divalent cations across the cell membrane. Under appropriate experimental conditions, divalent cation entry across the cell membrane can be monitored as the rate at which the intracellular fluorescence of divalent cation indicators is quenched by the addition of Mn2+ to the extracellular medium. We report that addition of vasopressin to fura-2-loaded glomerular mesangial cells in culture markedly accelerated the rate at which Mn2+ quenched fura-2 fluorescence at its Ca(2+)-insensitive wavelength in the presence of extracellular NaCl, but that this quench response was attenuated when Cl- was removed from the extracellular medium by equimolar substitution with impermeant anions (gluconate, methanesulfonate, acetate, lactate). Similarly, loss of agonist-induced quench also occurred when Cl- was substituted with gluconate in K(+)-containing media. Addition of the Cl- channel inhibitor, 5-nitro-2-(3-phenylpropylaminobenzoic acid) (NPPB), also inhibited Mn(2+)-induced quench of fura-2 fluorescence following vasopressin addition. In contrast, in the presence of gramicidin to provide an alternate conductance pathway to accompany divalent cation entry, agonist-dependent Mn2+ quench occurred even in the absence of extracellular Cl-, indicating that the requirement for Cl- was not the result of cotransport on a common transporter nor the result of Cl- serving as a necessary cofactor for divalent cation entry. A similar dependence on extracellular Cl- was observed for other Ca(2+)-mobilizing agonists such as endothelin, as well as the intracellular Ca2+ ATPase inhibitor, thapsigargin. Extracellular Cl- dependence for agonist-induced divalent cation entry was also reflected in a corresponding extracellular Cl- dependence for agonist-induced mesangial cell contraction. It has been previously shown by ourselves (Kremer et al., 1992a, Am. J. Physiol., 262:F668-F678) and others that agonist-stimulated calcium mobilization in mesangial cells is accompanied by inhibition of K+ conductance and increased Cl- conductance. Accordingly, we conclude that the current findings suggest that activation of Cl- conductance provides regulated charge compensation for receptor-mediated divalent cation entry in response to Ca(2+)-mobilizing vasoconstrictor agonists in mesangial cells.

Intracellular pH regulation in cultured rat astrocytes in CO2/HCO3(-)-containing media.

We studied the regulation of intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat astrocytes using microspectrofluorometry and the pH-sensitive fluorophore 2',7'-bis(carboxyethyl-)-5,6-carboxyfluorescein. Control pHi was 7.00 +/- 0.02 in HCO3(-)-containing solutions at an extracellular pH of 7.35. Addition of 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) or amiloride decreased pHi, as did removal of extracellular Na+, while removal of extracellular Cl- was followed by an increase in pHi. Following exposure to an acid transient induced by increasing the CO2 content from 5 to 15%, pHi rapidly returned to base line, with an average initial rate of recovery of 0.10 pH units min-1 (corresponding to a mean acid extrusion rate of 6.3 +/- 0.36 mmolo l-1 min-1). Regulation of pHi was impaired when either amiloride or DIDS was added or Cl- was removed. This inhibition was enhanced when both DIDS and amiloride were present, and pHi regulation was completely blocked in the absence of extracellular Na+. The rapid regulation of pHi normally seen following a transient alkalinisation was not inhibited by amiloride or removal of Na+, but was partially inhibited by DIDS and by the absence of extracellular Cl-. The results are compatible with the presence of at least three different pHi-regulating mechanisms: a Na+/H+ antiporter, a Na(+)-dependent HCO3-/Cl- exchanger (both regulating pHi during a transient acidification), and a passive Cl-/HCO3- exchanger (regulating pHi during transient alkalinisation). The results fail to provide firm evidence of the presence of an electrogenic Na+/HCO3- symporter.